The present invention relates to matrix metalloproteinase (MMP) inhibitors, especially collagenase inhibitors, and to their use in the manufacture of medicaments.
There are many different types of collagen found in the body and they, together with other extracellular matrix components, for example, elastin, gelatin, proteoglycan and fibronectin, make up a large proportion of the body""s extracellular tissue. Matrix metalloproteinases (MMPs) are enzymes that are involved in the degradation and denaturation of extracellular matrix components. Collagenases, for example, are matrix metalloproteinases that degrade or denature collagen.
A large number of different collagenases are known to exist. These include interstitial collagenases, type IV-specific collagenases and collagenolytic proteinases. Collagenases are generally specific for collagens which, in their full triple helix structure, are extremely resistant to other enzymes.
Other MMPs are involved in the degradation and denaturing of different extracellular matrix components, for example, elastin, gelatin and proteoglycan. Some MMPs are able to degrade or denature several different types of collagen and also other extracellular matrix components. For example, stromelysin degrades type IV collagen, which is found in basement membrane, and also has an effect on other extracellular matrix components such as elastin, fibronectin and cartilage proteoglycans.
There is a classification system for MMPs, see Nagase et al 1992. For example, MMP1 is a collagenase that is sometimes called xe2x80x9ccollagenasexe2x80x9d, MMP2 is a 72kD gelatinase, MMP3 is stromelysin and MMP9 is a 92kD gelatinase. The official designations are used herein.
Collagenases have been implicated in a number of diseases, for example, rheumatoid arthritis [Mullins, D. E. et al 1983], periodontal disease and epidermolysis bullosa, and it has been proposed to use MMP inhibitors in the treatment of such conditions.
U.S. Pat. No. 5,183,900, U.S. Pat. No. 5,189,178 and U.S. Pat. No. 5,114,953 describe the synthesis of N-[2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide, also known as GM6001, Galardin or Galardin-MPI (trade names), and other MMP inhibitors, and their use in the prevention and treatment of corneal ulceration. Treatment of corneal ulcers with peptide hydroxamic acid inhibitors has been found to assist in the healing of those ulcers. Further details of such uses are given in Schultz et al 1992.
Also described in the above-mentioned U.S. patent specifications is the use of collagenase inhibitors in situations where bacterial enzymes may be detrimental to tissue, for example, in bacterial ulceration.
Other collagenase inhibitors based on hydroxamic acid are disclosed in WO 90/05716, WO 90/05719 and WO 92/13831. Such collagenase inhibitors are disclosed as being used in the management of disease involving tissue degradation, particularly disease involving collagen breakdown, and/or the promotion of wound healing.
Other synthetic MMP inhibitors and, in particular collagenase inhibitors that have been developed include those described in EP-A-126,974 and EP-A-159,396 and in U.S. Pat. Nos. 4,599,361 and 4,743,587.
One inhibitor undergoing clinical trials is BB-94 also known as Batimastat (British Bio-technology Ltd.). Potential uses of BB-94 for the control of cancer metatasis are described in EP-A-276436. It has been proposed to use an oral formulation of BB-94 in the treatment of bone cancer.
The present invention is concerned with the contraction of tissues, for example, scars. Contraction of tissues comprising extracellular matrix components, especially of collagen-comprising tissues, may occur in connection with many different pathological conditions and with surgical or cosmetic procedures. Contracture, for example, of scars, may cause physical problems, which may lead to the need for medical treatment, or it may cause problems of a purely cosmetic nature.
It has been proposed that contraction is cell-mediated and a number of studies have suggested possible mechanisms for cell mediated collagen contraction [Gabbiani et al. 1972, Ehrlich and Rajaratnam 1990]. Investigations have been made into the role, if any, played by MMPs in the process of contracture. However, according to one proposition, MMPs are not produced during contraction [Schor et al. 1980]. According to another proposition, MMPs are produced during lattice contraction but are not implicated in the contractile process [Nakagawa et al. 1989, Mauch et al. 1989, Lambert 1992]. Instead, it has been proposed that contraction is dependent upon the extracellular lattice cell number, upon there being an intact actin cytoskeleton, and upon attachment of the cells to the extracellular matrix.
The present invention is based on the surprising observation that, during experiments on in vitro models of scar contraction, collagen (the main component of scar tissue) appears to be invaded and permanently remodelled by fibroblasts and that such invasion and remodelling is inhibited by collagenase inhibitors. The remodelling generally appears as contraction of the collagen, which contraction is inhibited by inhibition of collagenase. Furthermore, inhibition of other MMPs also results in inhibition of contraction. The observation that contraction of the tissue involves MMPs is particularly surprising since previous investigations have shown that MMPs are not produced during contraction while other investigations have indicated that MMPs are produced but are not involved in the contractile process (see above).
The present invention provides the use of an MMP inhibitor in the manufacture of a medicament for the treatment or prophylaxis of a natural or artificial tissue comprising extracellular matrix components to inhibit, i.e. restrict, hinder or prevent, contraction of the tissue, especially contraction resulting from a pathological condition or from surgical or cosmetic treatment.
The present invention also provides a method for the inhibition in vivo or in vitro of contraction of a natural or artificial tissue comprising extracellular matrix components, which comprises administering an MMP inhibitor to the tissue during and/or after its formation. Under in vivo condictions, a therapeutically effective amount of the MMP inhibitor should be administered.
The present invention especially provides the use of a collagenase inhibitor in the manufacture of a medicament for the treatment or prophylaxis of tissue comprising collagen to inhibit contraction of the tissue resulting from contraction of the collagen.
Further the present invention especially provides a method for the inhibition of contraction of tissue comprising collagen, resulting from contraction of the collagen, which comprises administering a therapeutically effective amount of a collagenase inhibitor to the tissue.
The methods of the present invention may be used for medical or cosmetic treatment.
The invention provides a method for the inhibition, for cosmetic reasons, of disfigurement caused by contraction of tissue comprising extracellular matrix components, which method comprises administering a matrix metalloproteinase inhibitor to the tissue.
Cosmetic treatments, such as chemical or physical dermal abrasion, used as anti-ageing treatments, cause trauma to the skin. Use of MMP inhibitors during the healing process which occurs after the initial abrasion is a cosmetic use of MMP inhibitors according to the present invention.
The present invention also provides the use of an MMP inhibitor to inhibit, i.e. restrict, hinder or prevent, invasion by cells, especially fibroblasts, into tissue comprising an extracellular matrix and/or migration by cells, especially fibroblasts, in or through tissue comprising an extracellular matrix.
The term xe2x80x9cMMP inhibitorxe2x80x9d is used herein to denote any substance that is capable of inhibiting, i.e. restricting, hindering or preventing, the action of an MMP. The term xe2x80x9ccollagenase inhibitorxe2x80x9d is used herein to denote any substance that is capable of inhibiting, i.e. restricting, hindering or preventing, the action of a collagenase. A collagenase inhibitor may be specific for one particular collagenase or may inhibit several different collagenases. A collagenase inhibitor may also inhibit other MMPs, in which case it may also be defined as an MMP inhibitor.
The term xe2x80x9cinhibitorxe2x80x9d as used herein includes agents that act indirectly by inhibiting the production of the relevant enzyme, for example an antisense molecule, as well as agents that act directly by inhibiting the enzyme activity of the relevant enzyme, such as, for example, a conventional inhibitor.
An MMP, e.g. collagenase, inhibitor may be naturally-occurring or synthetic. An MMP, e.g. collagenase, inhibitor may be an anti-MMP, e.g. anti-collagenase, antibody, either polyclonal or monoclonal. The inhibitory activity of a putative MMP inhibitor may be assessed by any method suitable for determining inhibitory activity of a compound with respect to an enzyme. Such methods are described in standard textbooks of biochemistry. A more detailed description of MMP, e.g. collagenase, inhibitors is given below.
Collagen is the major component of scar and other contracted tissue and as such is the most important structural component to consider. Nevertheless, scar and other contracted tissue also comprises other structural components, especially other extracellular matrix components, for example, elastin, which may also contribute to contraction of the tissue. MMPs are involved in the synthesis and degradation of such components. In general, a collagenase inhibitor is used as the MMP inhibitor in accordance with the present invention but, it may be appropriate to use instead an inhibitor of an MMP other than a collagenase. It may be particularly advantageous to use a combination of a collagenase inhibitor and one or more inhibitors of other MPs or to use an inhibitor which inhibits both a collagenase and at least one or more other MMPs.
The mechanism and control of contraction of tissues comprising extracellular matrix components, for example, collagen-comprising tissues, is still poorly understood. Some degree of contraction appears to be part of the healing process, but the trigger for contraction is not known. The involvement of MMPs in contraction of tissues, for example, scar tissue, and the utility of MMP inhibitors according to the present invention in the inhibition, i.e. prevention, restriction and hindering, of contraction has been confirmed by the following experimental data:
When fibroblasts in an in vitro collagen gel model of scar contraction are subjected to antiproliferative agents after contraction has occurred, there is no significant expansion of the collagen gel, that is to say, no relaxation of the contraction, even when the fibroblast cells have been killed and the supporting cytoskeleton of the cells removed. The remodelling of collagen leading to contraction therefore appears to result from activity of one or more enzymic systems of the fibroblasts.
Fibroblasts involved in the contraction of collagen produce greater amounts of matrix metalloproteinase mRNAs and proteins than do control fibroblasts. This is associated with cellular invasion of the collagen. Invasion of the collagen and contraction are inhibited by the use of inhibitors specific to those MMPs coded for by the mRNAs which are present at higher levels in cells involved in contraction than in control cells:
Quantitative competitive reverse transcriptase polymerase chain reaction (QCRT-PCR) technique [Tarnuzzer and Schultz 1994] was used to study the levels of MMP mRNA produced by human ocular fibroblasts in collagen Type I lattices undergoing contraction in comparison with human ocular fibroblasts in monolayer cultures. It was found that in the collagen lattices the fibroblasts produced more mRNA for collagenase (MMP1), 72 kD gelatinase (MMP2) and stromelysin (MMP3) but not for 92 kD gelatinase (MMP9) than did the control cells in the monolayer culture. Levels of mRNA for MMPs 1, 2 and 3 in the lattices were found to be greater than those in the monolayer cultures; over 100 times greater at certain times during the contraction process (see Example 4 below). Gelatin zymography [Heussen and Dowdle 1980] was used to analyse and compare production of the gelatinolytic components of the cells. It was found that gelatinolytic activity was increased compared to controls. It appeared, therefore, that the increased mRNA production resulted in increased protein production.
Antibodies against MMPs 1, 2, 3 and 9 were tested as contraction inhibitors. It was found that the antibodies against MMPs 1, 2 and 3 gave significant inhibition of the contraction of collagen gels by human ocular fibroblasts but that the antibody against MMP9 did not result in inhibition of contraction.
In the present specification the term xe2x80x9ccontraction of collagenxe2x80x9d includes not only shrinkage of collagen but also any remodelling of collagen that leads to contraction of the tissue comprising that collagen. It also includes contributions made by other components of the tissue, especially other extracellular matrix components, for example, elastin.
As indicated above, contraction of tissues comprising extracellular matrix components, especially of collagen-comprising tissues, may occur in connection with many different pathological conditions and with surgical or cosmetic procedures. Contracture may cause physical problems, which may lead to the need for medical treatment, or it may cause problems of a purely cosmetic nature. It is therefore very valuable to have medicaments capable of inhibiting, i.e. restricting, hindering or preventing, such contraction. Important uses of such medicaments are described below. It should be understood, however, that the uses according to the present invention are not restricted to the manufacture of medicaments, or to methods of treatment, medical or cosmetic, suitable for the conditions described below. The present invention also includes use in the manufacture of medicaments or in methods of treatment suitable for use in any case where contraction of tissue comprising extracellular matrix components resulting substantially from extracellular matrix component contraction is occurring or may occur.
Although the discussion below refers specifically to collagen contraction and the use of collagenase inhibitors, broad spectrum MMP inhibitors and/or inhibitors of MMPs other than collagenases may be used in inhibiting contraction of tissue comprising extra-cellular matrix components and the present invention is to be understood as including the use of such MMP inhibitors as an alternative to the use of collagenase-specific inhibitors in the treatment of tissue comprising extracellular matrix components. The present invention also includes the use of broad spectrum MMP inhibitors and/or inhibitors of MMPs other than collagenases in addition to the use of collagenase inhibitors, for example, in the treatment of tissue comprising extracellular components especially collagen comprising-tissue.
Contraction of collagen-comprising tissue, which may also comprise other extracellular matrix components, frequently occurs in the healing of burns. The burns may be chemical, thermal or radiation burns and may be of the eye, the surface of the skin or the skin and the underlying tissues. It may also be the case that there are burns on internal tissues, for example, caused by radiation treatment. Contraction of burnt tissues is often a problem and may lead to physical and/or cosmetic problems, for example, loss of movement and/or disfigurement. The present invention therefore includes the use of MMP inhibitors, for example, collagenase inhibitors, for example, in the form of a medicament, to inhibit contraction of the burnt tissue as it heals.
A further aspect of the present invention is the inhibition of the contraction of skin grafts. Skin grafts may be applied for a variety of reasons and may often undergo contraction after application. As with the healing of burnt tissues the contraction may lead to both physical and cosmetic problems. It is a particularly serious problem where many skin grafts are needed as, for example, in a serious burns case.
An associated area in which the medicaments and methods of the present invention are of great use is in the production of artificial skin. To make a true artificial skin it is necessary to have an epidermis made of epithelial cells (keratinocytes) and a dermis made of collagen populated with fibroblasts. It is important to have both types of cells because they signal and stimulate each other using growth factors. A major problem up until now has been that the collagen component of the artificial skin often contracts to less than one tenth of its original area when populated by fibroblasts. MMP inhibitors, for example, collagenase inhibitors,may be used to inhibit the contraction to such an extent that the artificial skin can be maintained at a practical size.
One area of particular interest is the use of MMP, e.g. collagenase,inhibitors to prevent or reduce contracture of scar tissue resulting from eye surgery. Glaucoma surgery to create new drainage channels often fails due to scarring and contraction of tissues. A method of preventing contraction of scar tissue formed in the eye, such as the application of a suitable agent, is therefore invaluable. Such an agent may also be used in the control of the contraction of scar tissue formed after corneal trauma or corneal surgery, for example laser or surgical treatment for myopia or refractive error in which contraction of tissues may lead to inaccurate results. It is also useful in cases where scar tissue is formed on/in the vitreous humor or the retina, for example, that which eventually causes blindness in some diabetics and that which is formed after detachment surgery, called proliferative vitreoretinopathy. Other uses include where scar tissue is formed in the orbit or on eye and eyelid muscles after squint, orbital or eyelid surgery, or thyroid eye disease and where scarring of the conjunctiva occurs as may happen after glaucoma surgery or in cicatricial disease, inflammatory disease, for example, pemphigoid, or infective disease, for example, trachoma. A further eye problem associated with the contraction of collagen-comprising tissues for which the methods and medicaments of the present invention may be used is the opacification and contracture of the lens capsule after cataract extraction.
Cicatricial contraction, contraction due to shrinkage of the fibrous tissue of a scar, is common. In some cases the scar may become a vicious cicatrix, a scar in which the contraction causes serious deformity. A patient""s stomach may be effectively separated into two separate chambers in an hour-glass contracture by the contraction of scar tissue formed when a stomach ulcer heals. Obstruction of passages and ducts, cicatricial stenosis, may occur due to the contraction of scar tissue. Contraction of blood vessels may be due to primary obstruction or surgical trauma, for example, after surgery or angioplasty. Stenosis of other hollow visci, for examples, ureters, may also occur. Problems may occur where any form of scarring takes place, whether resulting from accidental wounds or from surgery. Medicaments comprising MMP inhibitors, e.g. collagenase inhibitors, may be used wherever scar tissue is likely to be formed, is being formed or has been formed.
Conditions of the skin and tendons which involve contraction of callagen-comprising tissues include post-trauma conditions resulting from surgery or accidents, for example, hand or foot tendon injuries, post-graft conditions and pathological conditions, such as scleroderma, Dupuytren""s contracture and epidermolysis bullosa. Scarring and contraction of tissues in the eye may occur in various conditions, for example, the sequelae of retinal detachment or diabetic eye disease (as mentioned above). Contraction of the sockets found in the skull for the eyeballs and associated structures, including extra ocular muscles and eyelids, may occur if there is trauma or inflammatory damage. The tissues contract within the sockets causing a variety of problems including double vision and an unsightly appearance.
Although the above discussion relates in particular to humans, animals may exhibit the conditions described above or similar or analogous conditions. The present invention therefore also relates analogously to medicaments and methods for use in veterinary practice for the treatment and care of animals and especialy for use in the treatment and care of mammals.
The present invention provides a method of treating a human or other mammal to inhibit contraction of tissue comprising an extracellular matrix component, especially contraction associated with a chemical burn, a thermal burn or a radiation burn, a skin graft, a post-trauma condition resulting from surgery or an accident, glaucoma surgery, diabetes associated eye disease, scleroderma, Dupytren""s contracture, epidermolysis bullosa or a hand or foot tendon injury, which comprises administering to the human or other mammal a therapeutically effective amount of an MMP inhibitor.
It appears that MMP inhibitors, e.g. collagenase inhibitors, inhibit contraction tissues comprising extracellular components, for example, collagen, caused by cells such as fibroblasts but do not appear to be able to bring about significant reversal of such contraction. Accordingly, tissue which is being affected should generally be treated at the time when the contraction is occurring. Preferably treatment should take place as early as possible, advantageously as soon as, and most advantageously before, the first signs of contraction are observed. In treatments, conditions or healing processes where contraction of extracellular component, e.g. collagen, comprising tissue is common. MMP inhibitors, e.g. collagenase inhibitors, may be used as a routine prophylactic measure before any signs of contraction have actually been seen.
Since active contraction appears to be associated with active production of MMPs, the treatment used to prevent the contraction should be continued over at least the period during which contraction is likely to occur. This may often be quite a significant period of time, for example, several years or even longer. Contraction may still occur even after an initially open wound appears to have healed, for example, in patients with burns. Also conditions such as hand tendon contraction involve contraction even though there is no wound as such.
As indicated above, the present invention involves the use of MMP inhibitors, especially collagenase inhibitors.
Both natural and synthetic MMP inhibitors (inhibitors of enzyme activity), including collagenase inhibitors, are known. Naturally-occurring MMP inhibitors include xcex12-macroglobulin, which is the major collagenase inhibitor found in human blood [Eisen et al 1970]. Naturally occurring MMP inhibitors are also found in tissues. The presence of tissue inhibitors of MMPs has been observed in a variety of explants and in monolayer cultures of mammalian connective tissue cells [Vater et el 1979 and Stricklin and Wegus 1983]. Not only collagenase inhibitors but also inhibitors for other MMPs, for example, gelatinase and proteoglycanase are found. MMP inhibitors are generally unable to bind the inactive (zymogen) forms of the respective enzymes but complex readily with active forms [Murphy et al 1981]. Tissue MMP inhibitors are found, for example, in dermal fibroblasts, human lung, gingival, tendon and corneal fibroblasts, human osteoblasts, uterine smooth muscle cells, alveolar macrophages, amniotic fluid, plasma, serum and the xcex1-granule of human platelets [Stricklin and Wegus 1983; Welgus et al 1985; Welgus and Stricklin 1983; Bar-Sharvit et al 1985; Wooley et al; 1976; and Cooper et al 1985].
Synthetic collagenase inhibitors and inhibitors for other MMPs have been and are being developed. Compounds such as EDTA, cysteine, tetracycline and ascorbate are all inhibitors of collagenases but are relatively non-specific. As indicated above, synthetic inhibitors that have defined specificity for MMPs, including collagenase inhibitors, are described in the literature. For example, U.S. Pat. Nos. 5,183,900, 5,189,178 and 5,114,953 describe the synthesis of N-[2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide, also known as GM6001 or Galardin (trade name), and other MMP inhibitors. Other collagenase inhibitors based on hydroxamic acid are disclosed in WO 90/05716, WO 90/05719 and WO 92/13831. Further synthetic MMP inhibitors and in particular collagenase inhibitors that have been developed include those described in EP-A-126,974 and EP-A-159,396 and in U.S. Pat. Nos. 4,599,361 and 4,743,587.
Yet another inhibitor is BB-94, also known as Batimastat (British Bio-technology Ltd.), see for example, EP-A-276436. Disclosed in W090/05719 as having particularly strong collagenase inhibiting properties are (4-(N-hydroxyamino)-2R-isobutyl-3S-(thio-phenyl-thiomethyl)succinyl]-L-phenylalanine-N-methylamide (especially good) and [4-(N-hydroxyamino)-2R-isobutyl-3S-(thiomethyl)succinyl]-L-phenylalanine-N-methylamide and in WO90/05716 [4-(N-hydroxyamino)-2R-isobutylsuccinyl]-L-phenylalanine-N-(3-aminomethylpyridine) amide and [4-N-hydroxyamino)-2R-isobutyl-3S-methylsuccinyl]-L-phenylalanine-N-[4-(2-aminoethyl)-morpholino] amide.
The contents of the patent specifications and literature references mentioned herein are hereby incorporated by reference.
As indicated above, the properties of natural and synthetic collagen inhibitors may vary. Individual inhibitors often have different specificities and potencies. Some inhibitors are reversible, others are irreversible. In general the more potent an inhibitor""s inhibitory effects on a collagenase the better. For some uses an inhibitor specific to one particular collagenase may be required but generally a broad spectrum MMP inhibitor, for example, GM6001 (Galardin (trade name)), is preferred.
GM6001 (Galardin (trade name)) is a very potent MMP inhibitor that is effective against collagenase. It has the structure: 
A detailed account of its ability to inhibit human skin fibroblast collagenase, thermolysin and Pseudomonas aeruginosa elastase is given in Grobelny et al, 1992. Its inhibition constants with three types of MMPs are now calculated to be:
Preferred MMP inhibitors for use according to the present invention include GM6001 (Galardin) and those synthetic inhibitors described and referred to above. Preferred inhibitors include peptide hydroxamic acids or pharmaceutically acceptable derivatives thereof. Especially preferred are those compounds that are described and claimed in U.S. Pat. No. 5,189,178; U.S. Pat. No. 5,183,900 or U.S. Pat. No. 5,114,953 and that are collagenase inhibitors. Those with low Ki values, i.e. high pKi values are generally preferred. GM6001 (Galardin (trade name)) is an MMP inhibitor that is especially preferred because it is one of the most potent collagenase inhibitors known at present. However, for certain applications it may be preferable to use a less potent (weaker) inhibitor.
The preferred broad spectrum MMP inhibitor for use in accordance with the present invention is GM6001 (Galardin (trade name)). It is able to inhibit the action not only of collagenases but of other MMPs as well.
Also preferred are inhibitors that are capable of inhibiting MMPs 1, 2 and 3 (collagenase, 72 kD gelatinase and stromelysin, respectively). These may be used individually or in combination.
As indicated above, an anti-MMP polyclonal or monoclonal antibody, especially an anti-collagenase antibody, may be used as an inhibitor. An MMP antigen may be used in immunisation protocols to obtain polyclonal antisera immunospecific for that enzyme. The antigen may be a hapten derived from an MMP, especially from an active site region, or may be a full-length MMP or a fragment thereof. Using standard protocols and mammalian subjects, such as rabbits or mice, polyclonal antibodies may be obtained. Those may then be used as inhibitors. Monoclonal antibodies may be produced according to standard procedures, for example, using an appropriate MMP antigen, for example, a collagenase antigen.
Antibodies which are specific for a particular MMP may be made and the use of such specific inhibitors may be preferred under certain circumstances. For example, an antibody to MMP1, MMP2 or MMP3 (collagenase, 72 kD gelatinase or stromelysin respectively) or a mixture of two or more thereof may be used.
An alternative method of inhibiting the action of an MMP is to reduce the amount of the MMP by preventing its production. One method of preventing protein production is by the use of antisense nucleic acid molecules. An antisense molecule need not be large; 20 base pairs is often sufficient. If the molecule is small it should be able to enter the cells unaided but liposomes can be used to assist entry if required. An antisense molecule will usually be designed to attach to the MMP mRNA but may be designed to attach to the appropriate DNA during replication and transcription.
Although antibodies usually bind to proteins it is possible to produce antibodies which bind to nucleic acids. Accordingly, there may be used as an inhibitor according to the present invention an antibody that binds to the mRNA or the DNA of the selected MMP and hence hinders production of the MMP.
Reducing the production of an MMP has the advantage that it should be possible to use a smaller amount of inhibitor than is required for direct inhibition of MMP enzyme activity because each MMP mRNA molecule and the MMP DNA is responsible for the production of many MMP enzyme molecules.
Inhibitors for use according to the present invention must be able to be used in high enough concentrations and large enough doses to give adequate inhibition without being toxic to cells with which they come into contact.
For the treatment of some conditions it may be preferred to use a medicament containing a collagenase inhibitor and at least one other enzyme inhibitor. That additional inhibitor may also have collagenase inhibitory properties and/or it may have inhibitory properties for a different enzyme, for example, for a different MMP. If two or more inhibitors are used then the second and any additional inhibitor preferably has inhibitory properties for an enzyme other than a collagenase. Additional inhibitors may be, for example, inhibitors of other MMPs such as a gelatinase or a stromelysin, or inhibitors of other enzymes that break down tissue such as serine proteases, for example, a serine protease inhibitor such as aprotinin may be used.
Cytokines, for example, interleukin-1, in the environment of collagen-comprising tissue may stimulate collagenase production and so it may also be preferable to include an inhibitor of cytokines in the manufacture of medicaments or in methods of treatment according to the present invention.
Medicaments according to the present invention are generally provided in a pharmaceutical preparation form suitable for topical administration, for example, an emulsion, suspension, cream, lotion, ointment, drops, foam or gel. Such preparations are generally conventional formulations, for example, as described in standard text books of pharmaceutics such as the British Pharmacopoeia. Other suitable pharmaceutical forms for topical administration include dry powders, aerosols and sprays, which may be especially suitable for application to burns. Further suitable pharmaceutical preparation forms include those for administration by injection or infusion, for example, sterile parenteral solutions or suspensions, especially for administration directly into, or into the area of, the extracellular matrix component, e.g. collagen, comprising tissue, for example, by subconjunctival, subcutaneous, interpleural or intraperitoneal injection, and also slow release delivery systems, for example, liposome systems.
The invention especially provides a pharmaceutical preparation, (other than a preparation suitable for use in the eye), suitable for application to a wound, including an ulcer, a burn or skin graft comprising a matrix metalloproteinase inhibitor. Advantageously the preparation is in the form of a solution, suspension, cream, ointment, or gel, in which the MMP inhibitor is in a concentration of from 0.4 xcexcg/ml to 400 xcexcg/ml. The MMP inhibitor is preferably a collagenase inhibitor.
Oral formulations may also be used. These may be in the form of tablets, capsules, powders, granules, lozenges or liquid or gel preparations. Tablets may be coated by methods well known in normal pharmaceutical practice. Liquid formulations include syrups. Oral formulations may be used to treat directly conditions such as stomach ulcers and may also be used to treat conditions systemically.
The inhibitor(s) may be dissolved or dispersed in a diluent or carrier. The choice of carrier depends on the nature of the inhibitor, its solubility and other physical properties, and on the method and site of application. For example, only certain carriers are suitable for preparations for use in the eye.
Carriers include ethylene glycol, silver sulphadiazine cream and hypromellose. These may be used in creams and drops. An acetate buffer system may also be used. Further pharmaceutically suitable materials that may be incorporated in pharmaceutical preparations include absorption enhancers, pH regulators and buffers, osmolarity adjusters, emollients, dispersing agents, wetting agents, surfactants, thickeners, opacifiers, preservatives, stabilizers and antioxidants, foaming agents and flocculants, lubricants, colourants and fragrances (generally only in primarily cosmetic preparations).
Gels and liposomes may be the preferred delivery method when the inhibitor is an antisense molecule.
Preferably a medicament according to the present invention is applied directly to an open wound or is injected directly into the site of tissue contraction.
Suitable medicaments may, however, be applied to the skin surface where the tissue to be treated is below that surface, the active ingredient then being absorbed by and passing through the skin. Penetration enhancers are preferably incorporated in such medicaments.
Medicaments according to the present invention comprising MMP inhibitors, for example, collagenase inhibitors, for use in the inhibition of the contraction of tissues comprising extracellular matrix components, for example, collagen-comprising tissues, may contain further pharmaceutically active ingredients, for example antibiotics, antifungals, steroids, and further enzyme inhibitors, for example, serine protease inhibitors (as described above). Further components for certain indications include growth or healing promoters such as epidermal growth factor (EGF), fibronectin and aprotinin. As mentioned above cytokine inhibitors may also be included.
The inhibitors will generally be used in liquid and other non-solid formulations having concentrations of around 0.4 to 400 xcexcg/ml. In some cases, however, higher concentrations may be required. The total amount used and the dose administered will depend on the severity and area of the contraction, the condition causing it and the physical characteristics of the patient and the site and method of administration.
The following non-limiting Examples illustrate the invention. The Examples relate to experiments carried out in vitro; the teachings are directly applicable in vivo, for example, to the treatment of humans and mammals, for example, as described in the specification.
The Examples illustrate the use of MMP inhibitors in vitro models of scar contraction and of artificial skin. They also include experiments which illustrate the lack of toxicity to cells of MMP inhibitors and their effect on cell morphology. Further experiments investigate the levels of some MMP mRNA and of some MMPs present in cells during contraction.